Presence of zoonotic agents in engorged ticks and hedgehog faeces from Erinaceus europaeus in (sub) urban areas

European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.


Background
Hedgehogs are host to a wide variety of bacterial and protozoan pathogens [1][2][3], of which a number have become a matter of concern to public health. Since hedgehogs often dwell in (sub)urban areas, people who rescue or rehabilitate hedgehogs can be exposed to a variety of these pathogens by contact with hedgehogs, their excrements, and vectors. European hedgehogs (Erinaceus europaeus) are a reservoir host for Borrelia burgdorferi sensu lato (Lyme borreliosis), which is widely distributed in the Netherlands, and contributes to maintenance of the bacterium in an enzootic cycle [3,4]. In addition, it was proposed that European hedgehogs are a suitable reservoir host for Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans [2]. Both Borrelia genospecies and A. phagocytophilum are transmitted by ixodid ticks, such as Ixodes ricinus that feed on various hosts and I. hexagonus that feed predominantly on European hedgehogs [5]. All three life stages of these tick species can feed on humans [6].
In addition to vector-borne agents, hedgehogs are a potential reservoir for enteric bacteria (such as Salmonella and Campylobacter), and protozoan parasites (Giardia and Cryptosporidium), which may cause enteritis in humans, livestock, and pets [1,[7][8][9]. The primary transmission route to humans is believed to be food-borne, however, (indirect) contact with an animal reservoir can be an alternative source of infection [9,10]. For instance, a study carried out in Denmark reported that strains of Salmonella Enteritidis, isolated from European hedgehogs, belong to the same clonal lineage as strains isolated from infected humans [11].
In contrast, the zoonotic potential of some enteric protozoan parasites has not been fully recognized. Many studies designed to determine genetic groups of protozoan parasites in various hosts, suggest a limited zoonotic potential for Giardia, since strains isolated from people were infrequently found in animals [12,13]. Although zoonotic transmission of livestock-associated Cryptosporidium has frequently been described [14], the extent to which wildlife (e.g. hedgehogs) act as a source for Cryptosporidium infection in humans remains unclear.
Finally, little is known about the potential reservoir competence of the European hedgehog for other pathogens transmitted by ixodid ticks, such as Candidatus Neoehrlichia mikurensis, an agent of human neoehrlichiosis, and B. miyamotoi, a recently discovered agent belonging to the relapsing fever group. A number of studies detected Candidatus N. mikurensis in Northern whitebreasted hedgehog (Erinaceus roumanicus) tissue samples in Hungary, and in I. hexagonus feeding on hedgehogs and dogs in the Netherlands and Germany, respectively [15][16][17]. However, the role of European hedgehogs and their ectoparasites in maintenance of this pathogen in an enzootic cycle is unknown. Borrelia miyamotoi is present in questing I. ricinus in the Netherlands [18], however, it has never been investigated in I. hexagonus before.
In the current study, the presence of a number of zoonotic vector-borne and enteric bacteria and two protozoan parasites was investigated in engorged ticks, obtained from European hedgehogs and hedgehog faeces. In addition, the presence of extended-spectrum cephalosporin (ESC)-resistant E. coli was investigated in faeces, since ESC-resistant E. coli are found in many animal and environmental reservoirs.

Collection of Ixodes ticks and DNA extraction procedures
Ixodes hexagonus and I. ricinus ticks were collected from European hedgehogs, rehabilitating in a hedgehog shelter in the city of Naarden, and obtained via the Dutch Wildlife Health Centre (DWHC, Utrecht) in 2010, 2011, and 2012. All hedgehogs originated from five different provinces in the Netherlands: Flevoland, Gelderland, Noord-Holland, Utrecht, and Zuid-Holland. In addition, 15 I. hexagonus ticks were collected from dead hedgehogs near the city of Ede (province of Gelderland), and from a zoo in the city of Emmen (province of Drenthe) in 2014. Collected samples included ticks of both sexes and all developmental stages with a majority of adult female ticks.
DNA from partially engorged ticks was extracted with ammonium hydroxide as described previously [19]. DNA from fully engorged ticks was extracted using the Qiagen DNeasy Blood & Tissue Kit according to the manufacturer's protocol for the purification of total DNA from ticks (Qiagen, Venlo, the Netherlands).
Detection of tick-borne pathogens using qPCR, conventional PCR, and sequencing procedures Ticks were tested individually for presence of B. burgdorferi s.l., B. miyamotoi, A. phagocytophilum and Candidatus N. mikurensis using (q)PCR assays, followed by sequencing for species identification when necessary. For the detection of B. burgdorferi s.l., a duplex real-time PCR was used, based on the detection of fragments of ospA and flagellin genes [20]. A conventional PCR assay, targeting the 5S-23S intergenic region [(IGS) was performed, for Borrelia genospecies identification [21]. Both strands of PCR products were sequenced by BaseClear (Leiden, The Netherlands), using the same forward and reverse primers as in conventional PCR. Borrelia genospecies identification was determined by comparison of sequences to isolates in-house molecular databases (PMID: 23602839). For detection of B. miyamotoi, a real-time PCR assay was used that targets a region of the flagellin gene, specific for B. miyamotoi [18]. For detection of A. phagocytophilum and Candidatus N. mikurensis a single duplex real-time PCR assay was used that targets a region of the A. phagocytophilum major surface protein (msp2) gene [22], and a region specific for Candidatus N. mikurensis of the heat shock protein gene groEL [17]. Due to limitations of available DNA, not all ticks (n = 628) were tested for all pathogens. For numbers of ticks tested for each vector-borne pathogen, see Table 1.
Collection of hedgehog faeces, DNA extraction, detection of enteric pathogens, protozoan parasites, and antimicrobial resistance genes No ethical approval is required for the experimental methods used in this study. The hedgehog shelter has a permit for handling and rehabilitating hedgehogs by the State Secretary for Economic Affairs, Agriculture and Innovation, according article 75 of the Dutch ' Animal Health and Welfare Act'. Hedgehog faeces were collected from 90 hedgehogs, rehabilitating in the hedgehog shelter in the city of Naarden in April (n = 58) and October (n = 32) of 2013. Hedgehogs originated from five different provinces in the Netherlands, described before, and were brought to the shelter due to apparent sickness or injury. Hedgehog faecal material was examined for the presence of Campylobacter by standard microbiological methods, according to ISO/DIS 10272-1 [23]. Confirmation was based on typical microscopic appearance of suspect colonies on mCCDA plates, and by PCR in order to distinguish between C. coli, C. jejuni, C. lari and C. upsaliensis isolates [24].
Faecal samples were also tested for the presence of Salmonella according to Annex D of ISO 6579 [25], and Shiga toxin-producing E. coli according to ISO/TS 13136 [26]. After the presence of Salmonella was confirmed, serotyping was performed using the method of Grimont and Weill [27].
Expanded spectrum cephalosporin-resistant E. coli (ESC-resistant E. coli) were isolated by direct streaking of a loop (10 μl) of hedgehog faeces on Brilliance E. coli/ coliform Selective Agar (Oxoid), supplemented with 1 μg/ml cefotaxime (Sigma). Suspected ESC-resistant E. coli were phenotypically confirmed with a combination disc-diffusion test according to CLSI guidelines [28]. Cefotaxime and ceftazidime discs, with and without clavulanic acid, were used to identify ESBL-producing E. coli. A cefoxitin disc was used to detect isolates with an AmpC phenotype.
For detection of protozoan parasites, DNA was isolated from faecal samples using the High Pure PCR template DNA isolation kit from Roche (Almere, The Netherlands), according to the manufacturer's instructions. Detection of Giardia duodenalis, Cryptosporidium parvum, and C. hominis was performed using a multiplex real-time PCR [29]. Molecular typing of Cryptosporidium species was performed by sequencing an amplified fragment of the GP60 gene [30]. The assemblage of G. duodenalis was established using a PCR on marker 4E1-HP, specific for either assemblage A or B [31], which are associated with human infections.

Results and discussion
Regarding tick-borne pathogens, we detected B. burgdorferi s.l. in 14% (60/435) of I. hexagonus ticks and 28% (7/25) of I. ricinus ticks feeding on European hedgehogs (Table 1) Table 1). These findings are consistent with previous studies, which revealed the presence of the same Borrelia genospecies in ticks feeding on hedgehogs in Germany and Switzerland [3,4]. This suggests that the European hedgehog may be a reservoir host for B. burgdorferi s.l. also in the Netherlands as, and may influence local Lyme borreliosis risk.
In addition to B. burgdorferi s.l. genospecies, A. phagocytophilum was detected as well in Ixodes ticks feeding on European hedgehogs. DNA was detected in 27% (68/ 251) of I. hexagonus ticks and in 24% (6/25) of I. ricinus ticks ( Table 1). The relatively high prevalence of A. phagocytophilum found in the current study supports the idea, proposed by other researchers, that E. europaeus is a reservoir host for this pathogen [2,4,32].
Ixodes hexagonus as a nidicolous species, rarely bites humans and its direct epidemiological importance is unknown [5,6]. However, it seems to contribute to the circulation of both B. burgdorferi s.l., and A. phagocytophilum in nature [2,33]. In addition its predominant host, E. europaeus, may harbour all life stages of generalist I. ricinus ticks, which successfully infect hedgehogs with at least one major group of zoonotic agents: B. burgdorferi s.l. [34]. In certain habitats, hedgehogs may be the main host for I. ricinus ticks, which may acquire pathogens via either co-feeding or systemic transmission [35]. Subsequently, I. ricinus ticks may transmit a number of bacterial pathogens (e.g. A. phagocytophilum and B. burgdorfei s.l.) to other vertebrates as well, including humans. To our knowledge, this is the first study that has tested ticks feeding on hedgehogs for B. miyamotoi, a spirochete belonging to the relapsing fever group. The absence of this pathogen in I. hexagonus may indicate that this specialist tick species is not a competent vector, or that E. europaeus is not a competent host for B. miyamotoi. However, it was shown that 4% of questing I. ricinus ticks were positive for B. miyamotoi in the Netherlands [36]. Therefore, it is also possible that the number of investigated I. ricinus ticks in the current study was not sufficient to detect this bacterium.
Finally, no Candidatus N. mikurensis DNA was detected in either I. ricinus or I. hexagonus ticks feeding on E. europaeus. This finding is consistent with another study, in which this pathogen was also not detected in I. hexagonus feeding on Dutch hedgehogs [17]. Human and animal cases of Candidatus N. mikurensis infections have (as of yet) not been reported in the Netherlands, and the prevalence of this pathogen in questing I. ricinus ticks is relatively low [17]. Therefore, it is still unclear whether this pathogen may pose risk to public health in the Netherlands.
We detected Salmonella in 10% (9/90) of hedgehog faecal samples ( Table 2). Salmonellosis is a zoonosis that has already been associated with hedgehogs, including E. europaeus. Several studies reported at least three different serotypes in hedgehogs that are pathogenic to humans: Salmonella Tilene, Salmonella Typhymurium and Salmonella Enteritidis [1,11]. Three isolates obtained from these faecal samples were characterized as Salmonella enterica subsp. enterica serotype Enteritidis, which is a common serotype pathogenic to humans [37].
One faecal sample (1%) contained Campylobacter, which is the second most common food-borne bacterium worldwide [10]. Further genotyping revealed C. jejuni, which is recognized as one of the main causes of human gastroenteritis. A study in Denmark also reported the presence of C. jejuni in hedgehogs, which were rehabilitating in private homes, or fed in gardens [9]. No Shiga toxin-producing E.coli were detected in the faecal samples investigated.
In addition to zoonotic enteric bacteria, 11% (10/90) of hedgehog faecal samples were positive for Giardia species (Table 2). Giardia is a genus of flagellated protozoan parasites divided into eight major genetic groups (A-H) called assemblages, which slightly differ in morphology and may cause disease in diverse vertebrate hosts [13]. We detected G. duodenalis assemblage A in hedgehog faecal samples, which is responsible for human infections worldwide [12]. However, data regarding the presence of Giardia in hedgehogs are scarce, and until now no Giardia assemblages were found associated with these animals.
We detected Cryptosporidium species in 9% (8/90) of hedgehog faecal samples as well. Cryptosporidium is another genus of protozoan parasites of vertebrates, which causes enteric infections in humans. In this study, two genospecies, C. parvum (subtype: IIaA17G1R1 and IIcA5G3) and C. hominis (subtype: IbA10G2) were observed. These subtypes cause the majority of cryptosporidiosis in humans [14]. Cryptosporidium hominis subtype Ib is primarily transmitted anthroponotically and, to the best of our knowledge, it has never been detected in hedgehog faeces before. In addition, C. parvum subtype IIaA17G1R1 has never been detected in these animals either, but was described in calves, which may play a role in the transmission of human cryptosporidiosis [38]. Interestingly, subtype IIcA5G3, which is considered to be human specific, has been isolated from hedgehog faeces previously [7]. The presence of those subtypes in hedgehog faeces may indicate transmission of the pathogen within hedgehog populations, as suggested before [7].
Finally, viable ESC-resistant E. coli were detected in 71% (64/90) of hedgehog faecal samples (Table 2). AmpCproducing E. coli were only found in the samples collected in April, but at a high prevalence of 86% (50/58). A lower prevalence of 41% (13/32) of ESBL-producing E.coli was observed in samples collected in October. Only one isolate collected in April had the same phenotype. To the best of our knowledge this is the first description of ESC-resistant E. coli in hedgehogs. In the literature, an ESBL-producing isolate from hedgehog faeces was reported, however, this isolate was later identified as a Klebsiella pneumoniaea strain [39]. The relatively high prevalence of ESC-resistant E. coli in tested hedgehog faeces, especially in the April samples is intriguing. If this might pose a risk to humans, handling rehabilitating hedgehogs has to be investigated. It is very likely that these enteric pathogens, protozoan parasites, and ESC-resistant E. coli detected in hedgehog faecal samples were acquired by ingestion of contaminated materials found in the habitat of hedgehogs, and originates from other animals or humans (waste, food, etc.).